Exposure of several lines of cultured cells to Beta-adrenergic agonists leads to a rapid loss in catecholamine-sensitive adenylate cyclase activity (densensitization) followed by a slower loss of Beta-receptors (downregulation). During desensitization, the receptors appear to uncouple from the cyclase as measured by decreased agonist affinity. Beta-Recptors from control and desensitization cells were analyzed for activity by membrane/membrane fusion techniques. Adenylate cyclase activity in the donor membranes was inactivated; the membranes were then fused with membranes lacking Beta-receptors and assayed for Beta-adrenergic stimulated adenylate cyclase. When the donor membranes were from desensitized cells, there was less activity than when they were from control cells. Thus, desensitization appears to involve a change in the receptor that impairs its ability to couple to adenylate cyclase. Cycloheximide, chloroquine and monensin inhibited downregulation of Beta-receptors but not desensitization of adenylate cyclase. Exposure of rat glioma C6 cells to agents that elevate intracellular cyclic AMP (cholera toxin, dibutyryl cyclic AMP) also caused downregulation of the receptors but not desensitization. Similar results were obtained in murine Leydig tumor cells that have receptors for human chorionic gonadotropin (hCG). Cells exposed to hCG became desensitized followed by a biphasic loss of receptors. The first phase depended on occupancy of the receptors by hCG and correlated with internalization and degradation of the bound hormone. The second phase of receptor loss occurred 8 hrs. after exposing the cells to hCG, was independent of receptor occupancy and was mimicked by cyclic AMP. Downregulation of hCG receptors was blocked by cycloheximide, which did not prevent desensitization of hCG-stimulated adenylate cyclase. Thus, desensitization appears to be a hormone-mediated event that does not require protein synthesis whereas downregulation can be induced both by hormones and cyclic AMP and does require protein synthesis.